Hubert E. Nethercott

Expression of neurodevelopmental markers by cultured porcine neural precursor cells.

Despite the increasing importance of the pig as a large animal model, little is known about porcine neural precursor cells. To evaluate the markers expressed by these cells, brains were dissected from 60‐day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor, and platelet‐derived growth factor. Porcine neural precursors could be grown as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis using reverse transcription–polymerase chain reaction (RT‐PCR), immunocytochemistry, microarrays, and flow cytometry, and results compared with data from analogous human forebrain progenitor cells. Cultured porcine neural precursors widely expressed neural cell adhesion molecule (NCAM), polysialic acid (PSA)–NCAM, vimentin, Ki‐67, and Sox2. Minority subpopulations of cells expressed doublecortin, β‐III tubulin, synapsin I, glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) consistent with increased lineage restriction. A human microarray detected porcine transcripts for nogoA (RTN4) and stromal cell–derived factor 1 (SDF1), possibly cyclin D2 and Pbx1, but not CD133, Ki‐67, nestin, or nucleostemin. Subsequent RT‐PCR showed pig forebrain precursors to be positive for cyclin D2, nucleostemin, nogoA, Pbx1, vimentin, and a faint band for SDF1, whereas no signal was detected for CD133, fatty acid binding protein 7 (FABP7), or Ki‐67. Human forebrain progenitor cells were positive for all the genes mentioned. This study shows that porcine neural precursors share many characteristics with their human counterparts and, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling

Robust strategies for developing patient‐specific, human, induced pluripotent stem cell (iPSC)‐based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial‐to‐complete elimination of feeder layers, use of defined media, and single‐cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long‐term, single‐cell passaging of PSCs using a feeder‐free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large‐scale, cGMP‐compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

The Autism Spectrum Disorders Stem Cell Resource at Children’s Hospital of Orange County: Implications for Disease Modeling and Drug Discovery

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD‐specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action‐potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC‐based models of ASD‐associated diseases. The ASD Stem Cell Resource at the Childrens Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.

Traditional human embryonic stem cell culture.

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous culture for many years. hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs and have evolved considerably in the years since the first hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture - they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently using the characterization methods, such as karyotyping, immunocytochemistry, gene expression analysis, and flow cytometry, provided in this manual.

Cancer field effects in normal tissues revealed by Raman spectroscopy.

It has been demonstrated that the presence of cancer results in detectable changes to uninvolved tissues, collectively termed cancer field effects (CFE). In this study, we directly assessed the ability of Raman microspectroscopy to detect CFE via in-vitro study of organotypic tissue rafts approximating human skin. Raman spectra were measured from both epidermis and dermis after transfer of the rafts to dishes containing adherent cultures of either normal human fibroblasts or fibrosarcoma (HT1080) cells. Principal components analyses allowed discrimination between the groups with 86% classification accuracy in the epidermis and 94% in the dermis. These results encourage further study to evaluate the Raman capacity for detecting CFE as a possible tool for noninvasive screening for tumor presence.

Neural progenitor cells from an adult patient with fragile X syndrome.

BackgroundCurrently, there is no adequate animal model to study the detailed molecular biochemistry of fragile X syndrome, the leading heritable form of mental impairment. In this study, we sought to establish the use of immature neural cells derived from adult tissues as a novel model of fragile X syndrome that could be used to more fully understand the pathology of this neurogenetic disease.MethodsBy modifying published methods for the harvest of neural progenitor cells from the post-mortem human brain, neural cells were successfully harvested and grown from post-mortem brain tissue of a 25-year-old adult male with fragile X syndrome, and from brain tissue of a patient with no neurological disease.ResultsThe cultured fragile X cells displayed many of the characteristics of neural progenitor cells, including nestin and CD133 expression, as well as the biochemical hallmarks of fragile X syndrome, including CGG repeat expansion and a lack of FMRP expression.ConclusionThe successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents.

Derivation of Induced Pluripotent Stem Cells by Lentiviral Transduction

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.

Immunocytochemical analysis of human pluripotent stem cells.

This chapter will describe the most common immunocytochemical method utilized in the stem cell field - using fluorescently tagged secondary antibodies to detect a primary antibody that is bound to an epitope on a molecule of interest. Secondary antibodies recognize the heavy chain of the primary antibodys isotype. Generally, these methods employ an incubation period of the sample with the primary antibody, a series of washes to remove unbound primary antibody, a secondary incubation period of the sample with the fluorescently conjugated secondary antibody, followed by washes and preparation for microscopy.

Neural Progenitor Cell Culture

Publisher Summary Cultured neural progenitor cells hold considerable promise, both in terms of their application to a wide variety of research projects, and their use in development of therapeutic modalities. In the case of human neural progenitor cells (hNPCs), the primary source has been donated fetal tissue. However, the post-mortem brain could be a source of a vast supply of hNPCs that could reduce or eliminate reliance on fetal or embryonic sources. The practicality of such an approach is supported by recent work demonstrating the viability of hNPCs obtained from cadaveric donors, even after post-mortem intervals exceeding 20 h. The number of hNPCs with high proliferative and differentiation potentials, is greatest in the youngest brains, so brains harvested from premature and neonatal infants provide the best available post-natal, so post-mortem source of hNPCs. For example, well over 10 000 neonatal deaths with no neurological involvement occur annually in the USA (National Center for Health Statistics); thus, cells harvested from these patients may ultimately open up major new options for the prevention or repair of neurological disease or injury. This chapter describes the basic procedures to be used for the successful culture of human neural progenitor cells, including establishment of primary cultures, passaging, differentiation, and cryopreservation. It also describes the establishment of glial cultures, which are used to generate conditioned medium for differentiation of the neural progenitor cultures.

A novel, long-lived, and highly engraftable immunodeficient mouse model of mucopolysaccharidosis type I

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.